Many vaccines are currently being developed for human cancer immunotherapy and for treatment of infectious diseases, such as malaria, AIDS, hepatitis C virus, and SARS. Given the rapidity with which new emerging pathogens can appear, it is important to improve animal models that could be used to evaluate vaccination strategies and the protective capacity of different epitopes quickly and reliably. Furthermore, in vivo studies are already required to assess crucial variables of vaccine behavior that are not easily evaluated or impossible to measure in vitro, such as vaccine immunogenicity, vaccine formulation, route of administration, tissue distribution, and involvement of primary and secondary lymphoid organs. Because of their simplicity and flexibility, small animals, such as mice represent an attractive alternative to more cumbersome and expensive model systems, such as nonhuman primates, at least for initial vaccine development studies.
The moderate efficacy observed in several clinical trials of vaccines, which were found to be protective in wild-type animal studies (McMichael, A. J. & Hanke, T. Nat Med 9, 874-880 (2003)), may be partly explained by the different influence that human and animal MHC have on the outcome of the immune response, since animal MHC and human HLA molecules do not present the same optimal epitopes (Rotzschke, O. et al. Nature 348, 252-254 (1990)). Thus, despite some limitations, transgenic mice expressing human HLA should represent a useful improvement over wild-type mice as a preclinical model for testing vaccine candidates, evaluating the potential risk that the vaccines could induce autoimmune disorders, and devising better therapeutic strategies based on the human restriction element.
Cytotoxic T Cells
Cytotoxic T cells (CTL) play a crucial role in the eradication of infectious diseases and in some cases, cancer (P. Aichele, H. Hengartner, R. M. Zinkernagel and M. Schulz, J Exp Med 171 (1990), p. 1815; L. BenMohamed, H. Gras-Masse, A. Tartar, P. Daubersies, K Brahimi, M. Bossus, A. Thomas and P. Druhile, Eur J Immunol 27 (1997), p. 1242; D. J. Diamond, J. York, J. Sun, C. L. Wright and S. J. Forman, Blood 90 (1997), p. 1751). Recombinant protein vaccines do not reliably induce CTL responses (Habeshaw J A, Dalgleish A G, Bountiff L, Newell A L, Wilks, D, Walker L C, Manca F. 1990 November; 11(11): 418-25; Miller S B, Tse H, Rosenspire A J, King S R. Virology. 1992 December; 191 (2):9 73-7). The use of otherwise immunogenic vaccines consisting of attenuated pathogens in humans is hampered, in several important diseases, by overriding safety concerns. In the last few years, epitope-based approaches have been proposed as a possible strategy to develop novel prophylactic and immunotherapeutic vaccines (Melief C J, Offringa R, Toes R E, Kast W M. Curr Opin Immunol. 1996 October, 8(5):651-7; Chesnut R W, Design testing of peptide based cytotoxic T-cell mediated imrnunotherapeutic to treat infiction disease, cancer, in Ppowell, M F, Newman, M J (eds.): Vaccine Design: The Subunit, Adjuvant Approach, Plenum Press, New-York 1995, 847). This approach offers several advantages, including selection of naturally processed epitopes, which forces the immune system to focus on highly conserved and immunodominant epitopes of a pathogen (R. G. van der Most, A. Sette, C. Oseroff, J. Alexander, K. Murali-Krishna, L. L. Lau, S, Southwood, J. Sidney, R. W. Chesnut, M. Matioubian and R. Ahmed, J Immunol 157 (1996), p. 5543) and induction of multiepitopic responses to prevent escape by mutation such observed in HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. It also allows the elimination of suppressive T cell determinants, which might preferably elicit a TH2 response, in conditions where a TH1 responses is desirable, or vice-versa (Pfeiffer C, Murray J, Madri J, Bottomly K. Immunol Rev. 1991 October; 123:65-84; P Chaturvedi, Q Yu, S Southwood, A Sette, and B Singh Int Immunol 1996 8: 745-755). It finally provides the possibility to get rid of autoimmune T cell determinants in antigens, which might induce undesirable autoimmune diseases. Protective antiviral or anti-tumoral immunity using CTL epitope-peptides has been achieved in several experimental models (D. J. Diamond, J. York, J. Sun, C. L. Wright and S. J. Forman, Blood 90 1997, p. 1751; J. E. J. Blaney, E. Nobusawa, M. A. Brehm, R. H. Bonneau, L. M. Mylin, T. M. Fu, Y. Kawaoka and S. S. Tevethia, J Virol 72 (1998), p. 9567).
CTL epitope definition based on the usage of human lymphocytes might be misleading due to environmental and genetic heterogeneity that lead to incomplete results, and due to technical difficulties in isolating CTL clones. HLA class I or class II transgenic mice described to date have proved to be a valuable tool to overcome these limitations as illustrated by the identification with such animal models of novel CTL and T helper epitopes (Hill A V. Annu Rev Immunol. 1998; 16:593-617; Carmon L, EI-Shami K M, Paz A., Pascolo S, Tzehoval E, Tirosb B, Koren R, Feldman M, Fridkin M, Lemonnier F A, Eisenbach L. Int J Cancer, 2000 Feb. 1; 85(3):391-7). These mice have also been used to demonstrate: i) good correlation between peptide HLA binding affinity and immunogenicity (Lustgarten J, Theobald M, Labadie C, LaFace D, Peterson P, Disis M L, Cheaver M A, Sherman L A. Hum Immunol. 1997 February; 52(2):109-18; Bakker A B, van der Burg S H, Huijbens R J, DRijfhout J W, Melief C J, Adema G J, Figdor C G. Int J Cancer. 1997 Jan. 27; 70(3):302-9), ii) significant overlap between the murine and human CTL system at the level of antigen processing (same epitopes generated), and iii) comparable mobilization against most antigens of the CTL repertoires in HLA transgenic mice and humans (Wentworth, P. A., A. Vifiello, J. Sidney, E. Keogh, P, W. Chesnut, H. Grey, A. Sette. 1996. Eur. J. Immunol. 26:97; Alexander, J., C. Oserof, J. Sidney, P. Wentworth, E. Keogh, G. Hermanson, F. V. Chisari R. T, Kubo, H. M, Grey, A, Sette, 1997. J. Immunol. 159:4753).
To date, synthetic peptide-based CTL epitope vaccines have been developed as immunotherapeutics against a number of human diseases [18-20]. However, only moderate efficacy was observed in several clinical trials (21). This may be partly explained by the failure of these vaccines to induce sufficiently strong CTL responses. Indeed, recent reports suggest the need for CD4+ T-cell help to obtain maximum CTL response (A. J. Zajac, K. Murali-Krishna, J. N. Blattman and R. Ahmed, Curr Opin Immunol 10 (1998), p. 444; Firat H, Garcia-Pons F, Tourdot S, Pascolo S, Scardino A, Garcia Z, Michel M L, Jack R W, Jung O, Kosmatopoulos K, Mateo L, Suhrbier A, Lemonnier F A, Langlade-Dernoyen P Eur J Irmnunol 29, 3112, 1999).
CTL are critical components of protective immunity against viral infections, but the requirements for in vivo priming of CTL are not completely understood. It is now accepted that Th cells are usually essential for CTL priming with synthetic peptides. With respect to synthetic CTL epitopic peptides, several studies point to a mandatory need for Th lymphocyte stimulation to induce optimal CTL responses (C. Fayolle, E. Deriaud and C. Leclerc, J Immunol 147 (1991), p, 4069; C. Widmann, P. Romero, J. L. Maryanski, G. Corradin and D. Valmori, J Immunol Meth 155 (1992), p. 95; M. Shirai, C. D. Pendkton, J. Ahlers, T. Takeshita, M. Newman and J. A. Berzofsky, J Immunol 152 (1994), p. 549; J. P. Sauet, H. Gras-Masse, J. G. Guillet and E. Gomard, Int Immunol 8 (1996). p. 457). Several of these studies showed that activation of a CD8+ T cell requires simultaneous interaction of a CD4+ T helper cell and a CD8+ T cell with the same antigen-presenting cell presenting their cognate epitopes (Ridge J P, Di Rosa F, Matzinger P. Nature. 1998 Jun. 4; 3 93 (6684):474-8). The relevance of this three-cell interaction for priming of CTLs is confirmed by studies with viral epitopes, and animal models, since in vivo induction of CTLs was most efficient when CTL and Th epitopes were physically linked rather than administered as an unlinked mixture (Shirai M, Pendleton C D, Ahlers J, Takeshita T, Newman M, Berzohky J A. J Immunol. 1994 Jan. 15; 152(2): 549-56; Oseroff C, Sette A, Wentworth P, Celis E, Maewal A, Dahlberg C, Fikes J, Kubo R T, Chesnut R W, Grey B X Alexander J. Vaccine. 1998 May; 16(8): 823-33). The capacity of CTL and Th antigenic peptides to efficiently induce CTL responses has been demonstrated both in experimental models (C. Fayolle, E. Deriaud and C. Leclerc, J Immunol 147 (1991), p, 4069; C. Widmann, P. Romero, J. L. Maryanski, G. Corradin and D. Valmori, J Immunol Meth 155 (1992), p. 95) and in humans (A. Vitiello, G. Ishioka, H. M. Grey, R. Rose, P. Famess, R. LaFond, L. Yuan, F. V. Chisari, J. Furze and R. Bartholomeuz, J Clin Invest 95 (1995), p. 341; B. Livingston, C. Crimi, H. Grey, G. Ishioka, F. V. Chisari, J. Fikes, H. M. Grey, R. Chesnut and A. Sette, J Immunol 159 (1997), p. 1383). Moreover, a potent Th response plays an important role not only for optimal induction of CTL responses, but also for maintenance of CTL memory (E. A. Walter, P. D. Greenberg, M. J. Gilbert, R. J. Finch, K-S. Watanabe, E. D. Tbomas and S. R. Riddell, N Engl J Med 333 (1995), p. 1038; Riddell S R, Greenberg P D, In Thomas E D, Blume K G, Forman S J (eds): Hematopoietic Cell Transplantation, 2nd edn. Malden, Mass.: Blackwell Science Inc., 1999). Finally, it has long been documented that CD4+ T “helper” cells are crucial in coordinating cellular and humoral immune responses against exogenous antigens.
Recently, a transgenic (Tg) mouse that expresses both HLA-A*0201 class I and HLA-DR1 class II molecules was established (BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond D J, Hum, Immunol. 2000 August; 61(8):764-79). The authors reported that both HLA-A*0201 and HLA-DR1 transgenes are functional in vivo, that both MHC class I and class II molecules were utilized as restriction elements, and that the product of the HLA-DR1 transgene enhances the HLA-A*0201-restricted antigen-specific CTL responses (BenMohamed L, Krishnan R, Longmate J, Auge C, Low L, Primus J, Diamond D J, Hum, Immunol. 2000 August; 61(8):764-79).
It is noteworthy that these HLA-A*0201/DR1 Tg mice expressed their own MHC H-2 class I and class II molecules. Because HLA class I transgenic mice expressing endogenous mouse MHC class I genes preferentially and often exclusively develop H-2 restricted CTL response (C Barra, H Gournier, Z Garcia, P N Marche, E Jouvin-Marche, P Briand, P Fillipi, and F A Lemonnier J Immunol 1993 150: 3681-3689; Epstein H, Hardy F., May J S, Johnson M H, Holmes N. Eur J Immunol. 1989 September; 19(9):1575-83; Le A X; E J Bernhard, M J Holterman, S Strub, P Parham, E Lacy, and V H Engelhard J Immunol 1989 142: 13 66-1371; Vitiello A, Marchesini D, Furze J, Sherman L A, Chesnut R W., J Exp Med. 1991 Apr. 1; 173(4):100715), and HLA class II transgenic mice expressing endogenous mouse MHC class II genes fail to induce reliable HLA class II restricted antigen-specific responses (Nishimura Y, Iwanaga T, Inamitsu T, Yanagawa Y, Yasunami M, Kimura A, Hirokawa K, Sasazuki T., J Immunol 1990 Jul. 1; 145(1):353-60), these HLA-A*0201/DR1 Tg mice are of limited utility to assess human-specific responses to antigen.
However, in HLA class I transgenic H-2 class I knock-out mice, or HLA class II transgenic H-2 class II knock-out mice, only HLA-restricted CTL immune responses occur (Pascolo S, Bervas N, Ure J M, Smith A G, Lemonnier F A, Perarnau, B., J Exp Med. 1997 Jun. 16; 185(12). 2043-51; Madsen L, Labrecque N, Engberg J, Dierich A, Svejgaard A, Benoist C, Mathis D, Fugger L., Proc Natl Acad Sci USA—1999 Aug. 31; 96(18):10338-43). In fact, HLA-A2.1-transgenic H-2 class I-knock-out (KO) mice exhibit the ability to mount enhanced HLA-A2.1-restricted responses as compared to HLA-A2.1-transgenic mice that still express the endogenous murine H-2 class I molecules (Pascolo, S. et al. J Exp Med 185, 2043-2051 (1997); Ureta-Vidal, A., Firat, H., Perarnau, B. & Lemonnier, F. A. J Immunol 163, 2555-2560 (1999); Firat, H. et al., Int Immunol 14, 925-934 (2002); Rohrlich, P. S. et al., Int Immunol 15, 765-772 (2003)). The inventors have made similar observations with HLA-DR1-transgenic mice, depending on whether or not they are deficient in H-2 class II molecules (A. Pajot, unpublished results). Furthermore, in the absence of competition from murine MHC molecules, the HLA-A2.1-transgenic H-2 class I-KO or HLA-DRI-transgenic H-2 class II-KO mice generate only HLA-restricted immune responses (Pascolo, S. et al. J Exp Med 185, 2043-2051 (1997)) (A. Pajot, unpublished results), facilitating the monitoring of HLA-restricted CD8+ and CD4+ T cell responses. However, protective immune responses against pathogens, which often require collaboration between T helper and cytotoxic CD8+ T cells, cannot be studied in the single HLA class I- or HLA class II-transgenic mice, which do not allow the simultaneous assessment of HLA class I and II human responses in the same mouse.
Accordingly, there exists a need in the art for a convenient animal model system to test the immunogenicity of human vaccine candidates comprising constructs containing human CTL epitopes and, in some cases, with the inclusion of high potency CD4+ Th (helper T lymphocyte) epitopes to sustain antiviral and antitumoral CD8+ T-cell activity (A. J. Zajac, K. Murali-Krishna, J. N. Blattman and R. Ahmed, Curr Opin Immunol 10 (1998), p. 444; Firat H, Garcia-Pons F, Tourdot S, Pascolo S, Scardino A, Garcia Z, Michel M L, Jack R W, Jung O, Kosmatopoulos K, Mateo L, Suhrbier A, Lemonnier F A, Langlade-Dernoyen P, Eur J Irmnunol 29, 3112, 1999). There is also a need for a system that allows the simultaneous assessment of the mutual coordination between a CTL response, a TH response (in particular s TH1 or TH2 response), and, optionally, a humoral response.